DNA vaccines against human immunodeficiency virus type 1 in the past decade

This article reviews advances in the field of human immunodeficiency virus type 1 (HIV-1) and AIDS vaccine development over the last decade, with an emphasis on the DNA vaccination approach. Despite the discovery of HIV-1 and AIDS in humans nearly 20 years ago, there is no vaccine yet that can prevent HIV-1 infection. The focus has shifted toward developing vaccines that can control virus replication and disease progression by eliciting broadly cross-reactive T-cell responses. Among several approaches evaluated, the DNA-based modality has shown considerable promise in terms of its ability to elicit cellular immune responses in primate studies. Of great importance are efforts aimed at improvement of the potency of this modality in the clinic. The review discusses principles of DNA vaccine design and the various mechanisms of plasmid-encoded antigen presentation. The review also outlines current DNA-based vaccine strategies and vectors that have successfully been shown to control virus replication and slow disease progression in animal models. Finally, it lists recent strategies that have been developed as well as novel approaches under consideration to enhance the immunogenicity of plasmid-encoded HIV-1 antigen in various animal models.     

Synaptotagmin I and IV are differentially regulated in the brain by the recreational drug 3,4-methylenedioxymethamphetamine (MDMA)

3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is a widely abused drug. In brains of mice exposed to MDMA, we recently detected altered expression of several cDNAs and genes by using the differential display polymerase chain reaction (PCR) method. Expression of one such cDNA, which exhibited 98% sequence homology with the synaptic vesicle protein synaptotagmin IV, decreased 2 h after MDMA treatment. Herein, the effect of MDMA on expression of both synaptotagmin I and IV was studied in detail, since the two proteins are functionally interrelated. PCR analyses (semi-quantitative and real-time) confirmed that upon treatment with MDMA, expression of synaptotagmin IV decreased both in the midbrain and frontal cortex of mice. Decreases in the protein levels of synaptotagmin IV were confirmed by Western immunoblotting with anti-synaptotagmin IV antibodies. In contrast, the same exposure to MDMA increased expression of synaptotagmin I in the midbrain, a region rich in serotonergic neurons, but not in the frontal cortex. This differential expression was confirmed at the protein level with anti-synaptotagmin I antibodies. MDMA did not induce down- or up-regulation of synaptotagmin IV and I, respectively, in serotonin transporter knockout mice (-/-) that are not sensitive to MDMA. Therefore, psychoactive drugs, such as MDMA, appear to modulate expression of synaptic vesicle proteins, and possibly vesicle trafficking, in the brain.     

Spontaneous complete remission in acute lymphoblastic leukaemia

Case records of 78 patients of acute lymphoblastic leukemia have been reviewed. Complete remission occurred in seven cases following an episode of septicemia and supportive care.     

In vitro characterisation of two naturally occurring mutations in the thrombin-sensitive region of anticoagulant protein S

The molecular consequences of two naturally occurring mutations in the thrombin-sensitive region of protein S were investigated using a combination of recombinant protein expression, functional analysis and molecular modelling. Both mutations (R49H and R70S) have been found in thrombosis patients diagnosed as having type I protein S deficiency. Molecular modelling analysis suggested the R49H substitution not to disrupt the structure of thrombin-sensitive region, whereas the R70S substitution could affect the 3D structure mildly. To elucidate the molecular consequences of these substitutions experimentally, site directed mutagenesis of protein S cDNA and expression in mammalian cells created the two mutants. The secretion profiles and functional anticoagulant activities of the protein S mutants were characterised. Secretion of the R49H mutant was similar to that of wild type protein S, whereas the R70S mutant showed moderately decreased expression. Neither of the mutants showed any major functional defects as cofactors to activated protein C (APC) in an APTT-based assay or in degradation of factor Va. However, both mutants demonstrated decreased activity in a factor VIIIa degradation assay, which in addition to APC and protein S also included factor V as synergistic APC cofactor. In conclusion, the R49H substitution did not produce a quantitative abnormality in vitro, raising doubts as to whether it caused the type I deficiency. In contrast, the experimental data obtained for the R70S mutant agrees well with the observed type I deficiency. Our study illustrates that in vitro experimental characterisation together with computer-based structural analysis are useful tools in the analysis of the relationship between naturally occurring mutations and clinical phenotypes.     

Integrated Pharmacokinetic-Driven Approach to Screen Candidate Anticancer Drugs for Brain Tumor Chemotherapy

The goal of the study was to develop an effective screening strategy to select new agents for brain tumor chemotherapy from a series of low molecular weight anticancer agents [ON123x] by the combined use of in silico, in vitro cytotoxicity, and in vitro ADME profiling studies. The results of these studies were cast into a pipeline of tier 1 and tier 2 procedures that resulted in the identification of ON123300 as the lead compound. Of the 154 ON123xx compounds, 13 met tier 1 screening criteria based on physicochemical properties [i.e., MW < 450 Da, predicted log P between 2 and 3.5] and in vitro glioma cell cytotoxicity [i.e., IC50 < 10 μM] and were further tested in tier 2 assays. The tier 2 profiling studies consisted of metabolic stability, MDCK-MDR1 cell permeability and plasma and brain protein binding that were combined to globally assess whether favorable pharmacokinetic properties and brain penetration could be achieved in vivo. In vivo cassette dosing studies were conducted in mice for 12 compounds that permitted examination of in vitro/in vivo relationships that confirmed the suitability of the in vitro assays. A parameter derived from the in vitro assays accurately predicted the extent of drug accumulation in the brain based on the area under the drug concentration–time curve in brain measured in the cassette dosing study (r² = 0.920). Overall, the current studies demonstrated the value of an integrated pharmacokinetic-driven approach to identify potentially efficacious agents for brain tumor chemotherapy.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-012-9428-4) contains supplementary material, which is available to authorized users.KEY WORDS: brain tumor, CNS, drug development, pharmacokinetics, preclinical     

Repeated episodes of C5a-induced neutrophil influx do not result in pulmonary fibrosis

Multiple reactive oxygen species-induced epithelial injury by glucose, glucose oxidase, and lactoperoxidase instillation in the lung results in a progressive interstitial fibrosis. To test the hypothesis that multiple pulmonary inflammatory responses alone would not result in fibrosis, three sequential inflammatory reactions were produced at weekly intervals in hamster lungs via intratracheal instillation of human recombinant C5a. Numbers of neutrophils and total inflammatory cells in bronchoalveolar lavage (BALF) increased significantly at 24 h after each C5a treatment compared with saline controls. Neutrophils increased by 3-, 33-, and 34-fold compared with the corresponding controls at 24 h after the first, second, and third doses, respectively, but returned to control levels by six days postinstillation. LTB₄ levels increased by 24% and 20% compared with the corresponding controls at 24 h after the first and second doses but were not different from controls at other times. Hydroxyproline levels in treated animals did not differ significantly from control levels throughout the study. Protein levels were significantly increased at 24 h after the second and third doses and six days after the third dose compared with the corresponding controls. Occasional foci of neutrophils in alveolar spaces were observed at 24 h after each dose, but they decreased in frequency after six days. No foci of neutrophils were observed six days after the final dose, although some epithelial degeneration was observed by transmission electron microscopy. Our results indicate that pulmonary inflammation resulting from repeated influx of neutrophils in response to multiple instillations of C5a in the lung does not cause sufficient injury to result in pulmonary fibrosis.     

Immunohistology of oestrogen receptor and D5 antigen in breast cancer: correlation with oestrogen receptor content of adjacent cryostat sections assayed by radioligand binding and enzyme immunoassay.

Two monoclonal antibodies recognising epitopes associated with oestrogen receptor protein were evaluated against the assayable soluble oestrogen receptor concentration in a series of 149 breast carcinomas. One antibody (anti-ER) recognises the hormone binding unit of oestrogen receptor and gives nuclear staining; the other antibody (anti-D5) was raised to a component of soluble oestrogen receptor and gives cytoplasmic staining. To minimise variations attributable to tumour heterogeneity and sampling error immunohistology using the two monoclonal antibodies, radioligand binding assays, enzyme immunoassays, and quantitative histology were done on adjacent frozen sections. Thirty nine per cent, 48%, 54%, and 43% of the tumours were found to be oestrogen receptor positive by radioligand binding assay, anti-ER and anti-D5 immunohistology, and enzyme immunoassay, respectively. Strong correlations (p less than 0.0005) were found between anti-ER immunohistology and the radioligand binding assay. Only weak correlations were found between anti-D5 immunohistology and the results of other assay methods for oestrogen receptor. Nuclear staining of human breast cancers with the anti-ER monoclonal antibody thus seems to be an acceptable alternative to biochemical assays, with the additional advantage of showing intercellular and regional heterogeneity for oestrogen receptor content.     

Clinicopathological significance of intratumoural variations in elastosis grades and the oestrogen receptor status of human breast carcinomas

Cryostat sections from seventy-eight female breast carcinomas were assayed for oestrogen receptors by isoelectric focusing. Adjacent cryostat sections stained by Miller's elastic/van Gieson's method were graded for elastosis. Elastosis was similarly graded on near-equatorial paraffin sections from the same tumours. A positive correlation was obtained between elastosis in the near equatorial sections and oestrogen receptor positivity (p less than 0.0005), menstrual status (p less than 0.05) and parity (p less than 0.01) but no correlation was found between these factors and elastosis graded on cryostat sections from the more peripheral areas which had been selected for oestrogen receptor assay. These observations suggest that the central region of breast carcinomas, where connective tissue responses are fully developed, exhibits grades of elastosis with greater clinical significance. This may explain the conflicting published observations on the correlations between elastosis and oestrogen receptor status, which we believe are due to the lack of uniformity in tissue sampling. The possible implications of the absence of significant correlation between elastosis grades and tumour size, nodal status and disease-free interval are discussed.     

Enhancement of potency and efficacy of NADA by PKC-mediated phosphorylation of vanilloid receptor

The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 microM NADA is 5-10% of the current induced by 1 microM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( approximately 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (approximately 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a approximately 15 mV depolarization and initiated a train of action potentials compared with 1 microM NADA that produced a approximately 5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.